An increasing number of epidemiological studies suggest that intestinal microbes are not only central to maintaining host health, but also constitute etiological factors for the initiation and progression of diseases of the intestinal tract. However, the mechanisms by which microbes affect intestinal health remain largely unknown. Using a powerful genetic model organism, Drosophila melanogaster, we have recently shown that microbes not only alter intestinal stem cell (ISC) proliferation, but also modulate the relative proportions of differentiated cell types in the epithelium. Importantly, we found that pathogens promote an enteroendocrine (EE) fate while non- pathogenic microbes promote an enterocyte (EC) fate, suggesting that pathogenic and non-pathogenic microbes influence ISC lineage in an opposing manner. Based on these results, and from our previous studies, we hypothesize that gut microbes modulate immune signaling pathways in ISCs to influence their lineage decisions. To test this hypothesis, we propose the following specific aims: Aim 1: We will determine the microbial characteristics (immunogenicity, virulence/damage) that modulate the cellular composition of the gut epithelium in both Drosophila and murine enteroids. In parallel, we will determine the relative contributions of cell loss, ISC proliferation and differentiation to this process. Aim 2: We will characterize how a classical immune pathway, the Imd/Relish pathway (NF?B homologue), acts in Drosophila ISCs and murine enteroids to influence differentiation in response to gut microbes. We will first identify ISC-specific Relish target genes using a combination of cell type-specific transcriptomics and targeted DamID (TaDa). We will then analyze how the Imd/Relish pathway interacts with other gene networks known to control ISC differentiation. Our studies will therefore demonstrate a new role for Imd/Relish that goes beyond the control of immune effectors and provide mechanistic insight into how this pathway alters stem cell lineage and epithelial composition. Aim 3: We will investigate how activation of the Janus kinase (JAK)-signal transducer of activator (STAT) pathway triggers EE fate commitment in Drosophila ISCs and murine enteroids. We will identify direct and indirect target genes of STAT in order to characterize downstream mechanisms and delineate the impact that JAK-STAT signaling has on ISC differentiation. Finally, we aim to clarify how the interaction between the JAK-STAT and Imd/Relish pathways determines the opposite ISC fates produced in the gut by pathogenic and non-pathogenic microbes. Outcomes of this research will improve our understanding of how the microbiota alters intestinal homeostasis in health and disease and demonstrate that different gut microbes (pathogenic vs non-pathogenic) alter gut epithelial composition by differentially modulating ISC differentiation. We will also identify a new role for pathways classically defined as immune pathways in affecting ISC differentiation, providing new mechanistic insight into how ISCs respond to their microbial environment. The mechanistic principles identified in our study will therefore pave the way to a better understanding of diseases of gut origin and potentiate the development of new therapies.